This invention relates to a liquid chromatography column and more particularly to the removal of the spent media bed from and the charging of a fresh media bed into a chromatography column without disassembling the column.
Chromatography is a process of separation of the components of a mixture of chemical substances. The separation occurs by percolation of fluid through a body or bed of comminuted or porous rigid material, the various components being resolved by their selective retardation as they are transported through the bed by a moving fluid or buffer. A solution of the substances to be separated becomes the moving phase of the system passing through the interstices in the stationary or continuous phase which are finely divided particles in the form of a gel or slurry. The substances in the moving phase is poured into the top of a chromatography column filled with the finely divided material, i.e., the media, that can absorb differentially the substances to be separated. The particular material used for the media varies widely with the substances to be separated. As the solution percolates down the column the components are separated from the buffer fluid which generally is pumped back into the top of the column so as to again pass down through the bed as a carrier. The different substances as they travel down the column at different rates form bands of the different substances which are individually collected at the outlet.
As aforesaid the media of the continuous phase is a very fine particulate slurry or gel initially, but after it has been used for sometime it hardens and loses its effectiveness and becomes a dense mass. This spent media must then be removed from the column and replaced in the column with a fresh supply, i.e., the column must be repacked.
A chromatography column comprises a hollow vertically disposed cylindrical housing including a liquid dispensing section at the upper end and through which the buffer and substances to be separated are dispensed to the media bed, and a liquid collecting section at the lower end for collecting the substances and buffer individually. The media or bed through which the buffer fluid and mixture to be separated and purified percolates is located between these sections. The liquid dispensing section and liquid collecting section each include a respective plate and at least one, and generally both, of the plates are connected in an assembly with an axially movable plunger-like body positioned within the housing at the respective end. After the column is charged with the bed media the bodies are forced relatively to each other to compress and pressurize the media bed which has been poured into the column.
The known prior art methods for packing the bed of small diameter columns used for research and development, e.g., only a few inches in diameter, are fully disclosed in Sakamoto et al U.S. Pat. No. 5,021,162 dated Jun. 4, 1991. In the conventional liquid flow method applicable to columns of larger diameter used in production of useful products such as synthetic insulin, e.g., approximately two feet in diameter, the bed media slurry is poured into a reservoir which has been temporarily connected to the upper end of the chromatography column. The reservoir is then closed at the top and liquid is pumped under pressure through the reservoir and the column. Excess liquid floating above the bed is pumped from the reservoir and the reservoir is then removed. This leaves part of the bed over-flowing above the top of the column, and the column is closed after this excess bed media is removed. In that method, as with most of the other prior art proposals discussed in the aforesaid patent, the top of the column must be disconnected in order to charge or pack the bed and must then thereafter be reassembled.
As aforesaid, after the column has been in use for a time, the fine particle slurry gradually hardens into a dense mass. When this occurs the bed loses its effectiveness and thereafter must be removed from the column. The removal of spent slurry in the prior art requires that the bottom of the column be opened and that the plunger-like assemblies be disconnected from the cylindrical housing. Thereafter the hardened slurry dense mass must be broken up by chopping and the like and removed from the column. The time required to remove this spent slurry, the reassembly of the bottom plunger-like assembly to the housing, the recharging of the housing and the reassembly of the top plunger-like assembly is relatively substantial. In fact, the life of the bed during separation and purification production runs may be no greater than the maintenance time required for the removal of the spent bed and the recharging of the column. Clearly, if less time is required to remove the spent slurry and recharge the column, production costs for the process may be substantially reduced.